Chicken intestinal brush border epithelial cells contain a calmodulin binding protein of 110 kDa which has been shown to be a myosin I type molecule. There are probably three calmodulin molecules associated with each heavy chain. This myosin does not form filaments, but does have an actin-activated MgATPase activity and shares other properties of the more conventional two-headed myosin molecules. We have shown that it is capable of translocating actin filaments in an in vitro motility assay. Motility and actin-activation of the MgATPase activity is inhibited at high calcium concentrations due to a dissociation of a fraction of the calmodulin molecules. Re-addition of calmodulin restores motility and actin-activation of the MgATPase activity. Tropomyosin binding to the actin filaments also inhibits motility and the actin-activated MgATPase activity and decreases the affinity of actin for myosin. Interestingly, immunofluorescent data demonstrates that myosin I does not co-localize to tropomyosin-rich regions of tissue culture cells. New evidence suggests that the calcium-inhibited and tropomyosin-inhibited myosin I interacts only weakly with actin.